49.3 I-lm long tapering graduaily until distinct hyaline tail terminus 13.5 I-lm long ... I-lm s'amincissant régulièrement jusqu'à une partie hyaline terminale longue ...
Fundam. appl. NemalOl.) 1996,19 (6), 593-599
Description of Meloidogyne fallax n. sp. (Nematoda : Heteroderidae), a root-knot nematode from The Netherlands Gerrit
KARSSEN
Plant Pmteetion Service, Nematology Section, PO. Box 9102, 6700 He, Wageningen, The Netherlands.
Accepted for publication 6 November 1995.
Summary - A root-knot nematode, Meloidogyne fallax n. sp., is described and illusuated from tomato (Lycopersicon esculeruum Mill.) from The Netherlands; this new species is characterized by : female stylet dorsaily curved, 14.5 I-lm long, with rounded to uansversely ovoid knobs, slightly sloping posteriorly; perineal panern with a moderately high dorsal arch with coarse suiae, indistinct 1atera1lines and venual region oval to angular; male stylet 19.6 I-lm long, with large rounded knobs, set off from the shaft; male head region slightly set off, round raised labial disc and distinct lateral lips present; second-stage juvenile 403 I-lm long, tail 49.3 I-lm long tapering graduaily until distinct hyaline tail terminus 13.5 I-lm long with broadly rounded tip. So far, the known distribution in Europe is resuicted to a smail number of locations in the south-eastern part of The Netherlands.
Résumé - Description de Meloidogyne fallax n. sp. (Nematoda: Heteroderidae), un nématode galligè1l€ des PaysBas. - Un nématode galligène, lvfeloidogyne fallax n. sp., provenant de la tomate (Lycopersicon esculemum l'vW1.) aux Pays-Bas, est décrit et figuré; la nouvelle espèce est caractérisée par le stylet de la femelle long de 14.5 I-lm, incurvé dorsalement, avec des boutons basaux arrondis à ovoïdes uansversalement, légèrement inclinés postérieurement; la figure périnéale avec une arche dorsale modérément haute avec des suies épaisses, des lignes latérales indistinctes et une région venuale ovale à angulaire; le stylet du mâle long de 19.6 I-lm, avec de grands boutons basaux bien séparés de la partie cylindrique; la région céphalique du mâle légèrement séparée du reste du corps, avec un disque labial arrondi et des lèvres latérales distinctes; les juvéniles de deuxième stade longs de 403 I-lm avec une queue longue de 49.3 I-lm s'amincissant régulièrement jusqu'à une partie hyaline terminale longue de 13.5 I-lm et se terminant en une exuémité bien arrondie. Sa répartition en Europe est jusqu'à présent limitée à un petit nombre de sites dans le sud-est des Pays-Bas. Key-words : lvfeloidogyne fallax, morphology, root-knot nematode, taxonomy, The Netherlands.
In 1992 a field plot experiment was conducted near Baexem, The Netherlands, ta assess the host suitability of Meloidogyne ehitwoodi Golden el a!., 1980 on different crops. Zea mays L., a good host for M. ehitwoodi (O'Bannon el a!., 1982; Jepson, 1987) was recorded as a non- ta poor hosto A critical re-examination of secondstage juveniles, compared with M. ehitwoodi paratypes, indicated differences in body, tail and hyaline tail terminus length. In the autumn of the same year, the Baexem population was studied biochemically. An unique malate dehydrogenase (MDH) pattern was detected (called M. chitwoodi B-type), deviating from M. chitwoodi phenotypes (Karssen, 1994; van Meggelen el a!., 1994). A potato root-knot nematode survey in 1993 revealed seven other isozyme deviating populations (B-types) in the Baexem region. After a detailed study, of ail known B-type populations (Karssen, 1995), they were considered ta be morphologically and biologically different from M. chitwoodi Golden el al., 1980. The nematode also differs from other known root-knot nematodes. Because of these differences this nematode is here designated as a new species and described as Meloidogynefallax n. sp. The species epithet refers to the misleading morphological resemblance to M. chitwoodi. ISSN 1164-5571/96/06
g 4.00 1 \\ GaUlhier-Viliars - OR5TOM
Materials and Inethods
A culture of M. fallax n. sp., derived from infected black saisify (Scorzonera hispanica L.) from Baexem, was maintained on tomato (Lycopersicon esculemum Mill. cv. Moneymaker) in a greenhouse and regularly checked for purity with isozyme electrophoresis (Karssen el a!., 1995). This culture was used for ail morphologic and morphometric studies. Second-stage juveniles 02) and males were extracted by periodically rinsing fresh samples of infected roots in a spray mist chamber. Adult females were hand picked from infected tomata roots. For light microscope (LM) studies eggs, J2, and males were fixed at 70 oC, and mounted in T AF (Courtney el a!., 1955). Perineal patterns were cut from live young females in 45 % lactic acid and mounted in glycerin (Taylor & Netscher, 1974). Adult females were fixed in hot T AF, the head region was cut and mounted in T AF. Drawings were made with a drawing tube, and photographs and measurements were taken with a light microscope using differential interference contrast (DIC). For scanning electron microscope (SEM) studies males and females were fixed in 3 % glutaraldehyde buf593
G. Karssen
fered with 0.05 M phosphate buffer (pH 6.8) for 1.5 h and post-fixed with 2 % osmium tetroxide for 2 h at 22 oc. The specimens were dehydrated in a seven-graded series of ethanol, critical-point dried with carbon dioxide, and sputter coated with a layer of 20 nm goldpalladium (Wergin, 1981). The nematodes were examined with a JeolJSM 5200 scanning electron microscope operating at 15 kV accelerating voltage. For preparation of type material females, males and J2 were fixed in hot FP [11 ml formalin (40 % formaldehyde), 1 ml propionic acid, 88 ml distilled water] and processed by a rapid glycerin-ethanol method (Seinhorst, 1959). The specimens were mounted in desiccated glycerin on Cobb slides.
Meloidogynefallax n. sp.
=Meloidogyne chitwoodi B-type in Van Meggelen et al. (1994), Karssen (1995) (Figs 1-8) MEASUREMENTS
See Table 1. DESCRIPTION
Female: Body annulated, pearly white, globular to pear shaped, with slight posterior protuberance and distinct neck region projecting from the body axis at an angle of up to 90° to one side. Head region set off from body, marked with one or two annules. Head cap distinct but variable in shape; labial dise slightly elevated. Cephalic framework weakJy sclerotized; vestibule extension distinct. Stylet cone dorsally curved and shaft cylindrical; knobs large, rounded to transversely ovoid, slightly sloping posteriorly from the shaft. Excretory pore located between head end and metacorpus levels. One or two large vesicles and several smaller ones located along the lumen lining. Pharyngeal glands variable in size and shape. Perineal pattern ovoid to oval shaped, sometimes rectangular; dorsal arch ranging from low to moderately high, with coarse striae. Tail terminus indistinct without punctations. Phasmids small and difficult to observe. Perivulval area devoid of striae. Laterallines indistinct (LM), appearing as a weak indentation under SEM, increasing towards the tail terminus region and resulting in a relatively large area without striae. Ventral pattern region oval to angular shaped; striae moderately coarse. Male: Body vermiform, slightly tapering anteriorly, bluntly rounded posteriorly. Cuticle with distinct transverse striae. Lateral field with four incisures; outer bands irregularly areolated; a fifth broken longitudinal incisure is rarely present near mid-body. Head slightly set off, with a single post-labial annule (sometirnes called head region) usually partly subdivided by a transverse incisure. Labial dise rounded, elevated and fused with mediallips. Prestoma hexagonal in shape with six inner cephalic sensilla adjacent to the rim. Medial lips cres594
cent shaped with raised edges at lateral sides. Four cephalic sensil1a small and marked by cuticular depressions on the mediallips. Amphidial openings appear as elongated slits between labial dise and medium sized lateral lips. Cephalic framework moderately sclerotized, vestibule extension distinct. Stylet cone straight; shaft cylindrical; knobs large and rounded, set off from the shaft. Pharynx with slender procorpus, metacorpus oval shaped with pronounced valve. Ventrally overlapping pharyngeal gland lobe variable in length. Hemizonid, 2-3 fLm in length, two to four annules anterior to excretory pore. Testis usually long, monorchic, with reflexed or outstretched germinal zone. Tail short and twisted. Spicules slender, curved ventrally; gubernaculum slightly crescent shaped. Phasmids located anterior to cloaca. Second-stage Juveniles : Body moderately long, vermiform, tapering at both ends but posteriorly more than anteriorly. Body annules small but distinct. Lateral field with four incisures, not areolated. Head region truncate, slightly set off from body. Head cap low and narrower than head region. Cephalic framework weakly sclerotized, vestibule extension distinct. Stylet slender and moderately long, cone straight; shaft cylindrical; knobs distinct, rounded and set off from the shaft. Pharynx with faintly outlined procorpus and oval shaped metacorpus with distinct valve. Oesophageal gland lobe variable in length, overlapping intestine ventrally. Hemizonid distinct at the level of the excretory pore. Moderately sized tail, gradually tapering until hyaline tail terminus, with inflated proctodeum. Phasmids difficult to observe, small, slightly posterior to anus. A rounded hypodermis marks the anterior position of the smooth hyaline tail terminus; tail terminus ending in a broadly rounded tip. Terminus generally marked by faint cuticular constrictions. Eggs (n = 30) : Length 89.7-103.6 fLm (94.4 ± 3.39; SE = 0.62); width 34.1-44.2 fLm (38.9 ± 3.17; SE = 0.58); length/width ratio 2.1-2.9 (2.4± 0.19; SE= 0.04). TYPE MATERIAL
Holotype: Female on slide WT 3127, collection of Agricultural University, Wageningen, The Netherlands. Paratypes : Two female perineal patterns and heads, two males and five J2's deposited at each of the fol1owing nematode collections: Agricultural University, Wageningen, The Netherlands (WT 3128-3130); Instituut voor Dierkunde, Rijksuniversiteit, Gent, Belgium; Rothamsted Experimental Station, Harpenden, U.K. TYPE HOST, TYPE LOCALITY AND DISTRIBUTION
Described from roots of tomato (Lycopersicon esculentum MilL). The nematodes were originally derived from infected roots of black salsify (Scorzonera hispanica L. cv. Lange Jan) from arable land one mile north of Baexem, province of Limburg, The Netherlands. The known disFundam. appl. NemalOl.
Meloidogyne fallax n. sp.
Tabk 1. Morphometrics of Meloidogyne fallax n. sp. (n Character
Females
Males
= 30; ail measurements in f-lm). J2
Ch:uacter
Females
L
4913 ±74.9 1171 ±193.6 403.2 ±15.2 (404.1-720.3) (736.2-1520.1) (381.4-435.2)
Metacorpus valve width
94.4 ±3.4 (89.7-103.6)
Greatest body diant
361.6 ±57.7 (2562-464.1)
Excretory pore-am. end
22.5 ±5.3 (12.6-32.9)
30.6± 2.1 (272-43.8)
14.3 ±0.7 (13.3-16.4)
Body diillD. at stylet knobs
17.7±0.6 (16.4-19.0)
Tai]
Body diillD. at excr. pore
26.2 ±\.7 (23.4-29.7)
Tai] terminus length 104±0.4 (9.5-10.7)
Body diillD. at anus
Spicule
Head region diam.
10.7 ±0.7 (9.5-12.0)
5.5 ±0.3 (5.1-6.3)
Gubernaculum
Neck diam.
97.7± 23.6 (64.6-160.2)
Stylet
14.5±0.4 (13.9-15.2)
10.8 ±0.4 (10.l-11.4)
Vulva-anus distance
15.9 ±1.8 (12.6-19.0) 1.4±0.3 (0.9-2.0)
10.1-0.5 (9.5-12.0)
Stylet shaft and knobs
8.9 ±0.5 (8.2-9.5)
55 ±0.4 (5.1-6.3)
c'
2.3 ±0.3 (2.0-2.5)
3.0 ±0.3 (2.5-3.2)
1.5±0.3 (13-1.9)
T
4.2 ±0.3 (3.8-4.4)
4.9 ±0.4 (3.8-5.1)
2.3 ±0.3 (1.9-2.5)
Body lengthlneck length
4.3 ±0.5 (3.8-6.3)
4.4 ±0.7 (3.2-5.7)
3.5 ±0.3 (3.2-3.8)
65.4±4.1 (58.8-72.7)
48.0 ±3.5 (44.2-54.4)
Stylet knob width DGO Am. end to metacorpus
496.5 ±144.1 (316.3-695.2) 24.7 ±1.8 (20.2-28.4)
Stylet cone
Stylet knob height
8.4 ±0.7 (6.4-9.3)
Metacorpus diam.
39.6±3.8 (31.6-44.9)
Metacorpus lengthlwidth
11±0.1 (0.9-1.2)
Metacorpus valve length
12.0±0.9 (10.1-13.9)
Vol. 19, n° 6 - 1996
8.2 ±05 (6.9-8.6)
Body lengthlant end to melllcorpus valve 1.9±0.2 (1.5-2.2)
M. fa/lax n. sp. is characterized by a dorsally curved
127.8 ±28.5 (82.7-20\.7)
3.3 ±0.9 (1.9-5.6)
Stylet knob \\~dth/height
DIAGNOSIS Ai'lD RELATIONSHIP
28.1 ±I.7 (23.8-40.4)
42.4 ±8.4 (24.4-62.1)
41.9 ±3.3 (34.8-47.4)
tribution of M. fallax n. sp. is restricted to the southeastern part of The Netherlands close to the Belgian and German border region (Fig. 9). The distribution of M. fa/lax n. sp. occurs adjacent to the M. chùwoodi dis tribution pattern, suggesting a parapatrie distribution.
38.2±6.8 (21.2-53.5)
4.8±0.3 (4.3-5.3)
Metacorpus length
4.0 ±0.3 (3.2-3.8)
49.3 ±2.2 (46.1-55.6)
7.7 ±0.5 (7.0-8.5)
Vulva slit length
14.6±0.7 (13.9-16.4)
Stylet base-ant end
9.2 ±1.4 (7.6-12.1)
13.5 ±1.0 (12.2-15.8)
Testis
19.6 ±0.8 (18.9-20.9)
69.1 ±3.4 (63.2-77.1)
266 ±20 (22.1-29.7)
2.7± 0.4 (1.9-3.2)
149.7 ±33.0 (96.4-224.6)
1209±11.4 (94.8-139.9)
11.7± 1.5 (9.5-15.2)
4.6± 0.3 (4.4-5.1)
Excretory poreiL x 100
J2 3.3 ±0.2 (3.2-3.8)
Phasmids-post. end
Head region height
Neck length
Males
1.6± 0.2 (1.4-2.0)
10.3 ±1.9 (8.3-12.9)
17.2±1.1 (16.1-19.3)
female stylet 14.5 !-Lm (13.9-15.2) long with rounded set off stylet knobs. Oval shaped perineal pattern with coarse striae and moderately high dorsal arch. Male stylet length 19.6 fJ.m (18.9-20.9) with prominent set off rounded knobs. The labial dise is elevated, crescem shaped medial bps raised at lateral side and distinct laterallips. TheJ2's hemizonid is at the same level with the excretory pore. Tail and hyaline tail length 49.3 fJ.m 595
G. Karssen
Fig. 1. Meloidogyne fallax n. sp. females. A : Pharyngeal region (laleral view); B, C: Slylels (laleml view); D-K: Female body shapes.
Fig. 3. SEM pholOgraphs of Meloidogyne fallax n. sp. fenlales. A-D : Perineal pallerns. (Scale bat = 10 f-lm).
c
A
Fig. 2. LM pholOgraphs offemales of Meloidogyne falJax n. sp. (A-D) and M. chitwoodi (E-F). A, B, D, E: Perineal pallerns; C, F: Head end (laierai view). (Scale bats: A, B, D, E = 25 f-lm; C, F= IOf-lm).
(46.1-55.6) and 13.5 fJ-m (12.1-15.8), respectively. M. fallax n. sp. reproduces by facultative meiotic parthenogenesis, the haploid chromosome number is n = 18 (H. v.d. Beek, pers. comm.). M.fallax n. sp. is characrerized by an unique malate dehydrogenase (MDH) pattern, not described by Esbenshade and Triamaphyllou (1987), and the lack of any major esterase (EST) band (Fig. 8). In combination these patterns are useful to dif596
F
o
E
G
Fig. 4. Meloidogyne fallax n. sp. males. A : Pharyngeal region (laierai view); B : Head end (laierai view); C-E; Slylels (laleral, laleral, ven/rai view, respecLively); F: Spicule and gubernaculum (laierai view); G: Laleral/ield al mid-body.
Fundam. appl. Nemalol.
Meloidogyne fallax n. sp.
Fig. 5. SEM (A-D) and LM (E-F) pholographs of males of Meloidogyne fallax n. sp. (A, B, D, E) and M. chirwoodi (C, F). A : Cephalic region (face view); B, C: Cephalic region (lateral vzéw); D: Tail (laierai view); E, F: Cephalic region (lateral view). (Scale bars: A, B, C = 1 !-lm; D = 5 !-lm; E, F = 10 !-lm).
B
=
G
A-f
Fig. 7. LM pholographs of second-stage juveniles of Meloidogyne fallax n. sp. (A-C, E, F) and M. chirwoodi (D) (laleral view). A-D : Tail variation (arrow = anus); E : Head end; F : Mela- and poStc01puS region (alTOW hemizonid). (Scale bar 10 !-lm).
=
Il
20J,lm G
:3
+ .~
Î~
.1) l"'~ .... ·1\ .......
.;. 0
0
(JO.
.
Fig. 8. Esterase (A-B) and malale dehydrogenase (C-D) isozyrne pauerns of Meloidogyne fallax n. sp. (A, C) and M. chirwoodi (B, D) (see also van Meggelen et al., 1994).
oU
O~).
